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脓毒症作为机体对感染反应失调的器官功能障碍综合征,常累及肾脏,是危重患者发生急性肾损伤最常见的原因之一。脓毒症引起的急性肾损伤不仅使住院死亡率增加6~8倍,还与远期慢性肾脏病的发展和远期死亡风险增加有关[1-3]。通常认为脓毒症相关急性肾损伤的发病过程与过度炎症反应、血流动力学障碍、凝血功能障碍、小管上皮细胞损害等有关[1],然而,脓毒症相关急性肾损伤的机制尚未完全阐明。近年来,代谢组学作为反应疾病内环境与内源性代谢物之间关系的一种方法,被广泛应用于各种疾病,因此,利用代谢组学探究脓毒症相关急性肾损伤中内源性代谢物的变化有助于进一步理解其发病机制。黄连作为常见中药,具有抗炎、抗氧化等功能,被广泛用于各种疾病[4]。既往研究发现黄连可以通过减轻炎症改善脓毒症诱导的急性肝损伤[5],而黄连是否对脓毒症相关急性肾损伤存在保护作用还有待进一步研究。本研究旨在研究黄连对脓毒症相关肾损伤的影响,并利用代谢组学探讨其潜在机制。
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假手术组、模型组及给药组Scr水平分别为(9.83±1.95)、(50.83±13.53)、(29.67±4.96)μmol/L;BUN水平分别为(8.08±0.84)、(27.67±5.22)、(16.33±2.69)mmol/L。模型组较假手术组Scr水平、BUN水平均上调;而给药组较模型组均下调,差异均有统计学意义(P<0.05,表1),表明黄连提取物在脓毒症相关急性肾损伤中可改善肾功能。
表 1 黄连提取物对小鼠外周血生化指标的影响
组别 Scr (μmol/L) BUN (mmol/L) 假手术组 9.83±1.95 8.08±0.84 模型组 50.83±13.53* 27.67±5.22* 给药组 29.67±4.96# 16.33±2.69# *P<0.05,与假手术组比较;#P<0.05,与给药组比较。 -
按照上述色谱-质谱条件,分别对各组样品进样分析,得到典型的总离子流图(图1)。
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采用PCA方法对各组样本进行整体分析,并通过质量控制样本的聚集程度对系统的稳定性进行考察。根据整体PCA得分图(图2)所示,质量控制样本均聚集良好,其离散度明显低于待分析样本的离散度,表明系统稳定性良好。
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采用PLS-DA方法对假手术组、模型组、给药组样本进行分析。首先对假手术组和模型组单独进行分析,两组在正/负离子模式下的得分图显示,假手术组与模型组区分明显(图3)。进一步对3组样本进行分析,3组在正/负离子模式下的得分图显示,假手术组与模型组有较明显的区分趋势,同时,给药组较模型组有一定程度的回调(图4)。提示脓毒症相关急性肾损伤中,经黄连提取物干预后,代谢物变化发生回调。
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S-plot可以很好地反映各个离子对组间差异的贡献程度,离原点越远的点表明其对组间差异的贡献度越大,其VIP值也越大。正离子模式下对照组和模型组的S-plot显示两组间存在差异代谢物(图5)。
在VIP值>1,且3组ANOVA分析以及Turkey两两分析P值均<0.05的条件下,共筛选并鉴别出16个差异代谢物,包括天冬氨酸、甘氨酸、苏氨酸、脯氨酸、苯丙氨酸、缬氨酸、酪氨酸、色氨酸、磷酸、苹果酸、柠檬酸、果糖、葡萄糖、松二糖、肌醇,主要参与氨基酸代谢和糖代谢(表2)。在这16个代谢物中,其中8个代谢物水平在黄连提取物干预下发生回调,它们是天冬氨酸、甘氨酸、苏氨酸、苯丙氨酸、酪氨酸、柠檬酸、葡萄糖、肌醇。提示黄连提取物可能通过改善脓毒症相关急性肾损伤的代谢物变化从而起到治疗作用。
表 2 脓毒症相关肾损伤差异代谢物及代谢途径
序号 保留时间(t/min) VIP 代谢途径 代谢物 分子式 倍数变化 模型组/假手术组 给药组/模型组 1 7.37 1.37 氨基酸代谢 L-天冬氨酸(L-aspartic acid) C4H7NO4 0.63 1.56 2 10.99 1.36 L-甘氨酸(glycine) C2H5NO2 1.25 0.78 3 12.65 1.49 L-苏氨酸(L-threonine) C4H9NO3 1.18 0.64 4 14.96 2.71 L-脯氨酸(L-proline) C5H9NO2 0.79 0.38 5 15.64 1.48 L-苯丙氨酸(L-phenylalanine) C9H11NO2 1.45 0.18 6 20.52 1.68 L-缬氨酸(L-valine) C5H11NO2 0.84 0.13 7 21.11 1.07 L-酪氨酸(L-tyrosine) C9H11NO3 1.28 0.12 8 25.58 1.93 色氨酸(tryptophan) C11H12N2O2 0.71 0.61 9 10.63 1.69 糖代谢 磷酸(phosphoric acid) H3PO4 0.7 0.99 10 14.13 1.08 L-苹果酸(L-malic acid) C4H6O5 0.9 0.64 11 17.03 1.56 L-苏糖酸(L-threonic acid) C4H8O5 0.81 0.37 12 20.1 1.63 柠檬酸(citric acid) C6H8O7 0.77 1.99 13 20.91 4.44 D-果糖(D-fructose) C7H15NO6 0.82 0.68 14 21.38 1.34 D-葡萄糖(D-glucose) C6H12O6 1.33 0.68 15 22.3 1.91 松二糖(turanose) C12H22O11 0.81 0.66 16 24.27 2.26 肌醇(inositol) C6H12O6 1.34 0.59
The protective effect of Rhizoma Coptidis extracts against the sepsis associated with acute kidney injury based on metabolic analysis
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摘要:
目的 探讨黄连提取物(Rhizoma Coptidis extracts,RCE)对脓毒症相关急性肾损伤的影响及潜在机制。 方法 将C57BL/6小鼠分为假手术组、模型组以及治疗组3组;采用试剂盒检测血清肌酐(serum creatinine,Scr)及尿素氮(blood urea nitrogen,BUN)水平;采用气相色谱-质谱进行代谢组学分析。 结果 模型组较假手术组Scr、BUN水平均上调;而治疗组较模型组均下调,差异均有统计学意义(P<0.05)。代谢组学分析共获得16个代谢物可能与脓毒症相关急性肾损伤过程有关,主要参与氨基酸代谢和糖代谢。在这16个代谢物中,8个代谢物水平在黄连提取物干预下发生回调。 结论 黄连提取物可能通过改善脓毒症相关急性肾损伤的代谢物变化从而起到治疗作用。 Abstract:Objective To investigate the potential mechanism of Rhizoma Coptidis extracts (RCE) against sepsis associated with acute kidney injury. Methods C57BL/6 mice were divided into sham group, model group and RCE treatment group. The levels of Scr and BUN were measured by test kits. Gas chromatography-mass spectrometry was used to analyze metabolic changes in kidneys. Results The levels of Scr and BUN were increased in the model group than sham, which were reversed by RCE. 16 metabolites related to the progress of sepsis associated with acute kidney injury were detected, which were involved in amino acid metabolism and carbohydrate metabolism. Among these metabolites, the level of 8 metabolites can be reversed with RCE treatment. Conclusion RCE might exert therapeutic effects in sepsis associated with acute kidney injury by altering multiple metabolic pathways. -
Key words:
- sepsis /
- acute kidney injury /
- Rhizoma Coptidis /
- metabonomics
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表 1 黄连提取物对小鼠外周血生化指标的影响
组别 Scr (μmol/L) BUN (mmol/L) 假手术组 9.83±1.95 8.08±0.84 模型组 50.83±13.53* 27.67±5.22* 给药组 29.67±4.96# 16.33±2.69# *P<0.05,与假手术组比较;#P<0.05,与给药组比较。 表 2 脓毒症相关肾损伤差异代谢物及代谢途径
序号 保留时间(t/min) VIP 代谢途径 代谢物 分子式 倍数变化 模型组/假手术组 给药组/模型组 1 7.37 1.37 氨基酸代谢 L-天冬氨酸(L-aspartic acid) C4H7NO4 0.63 1.56 2 10.99 1.36 L-甘氨酸(glycine) C2H5NO2 1.25 0.78 3 12.65 1.49 L-苏氨酸(L-threonine) C4H9NO3 1.18 0.64 4 14.96 2.71 L-脯氨酸(L-proline) C5H9NO2 0.79 0.38 5 15.64 1.48 L-苯丙氨酸(L-phenylalanine) C9H11NO2 1.45 0.18 6 20.52 1.68 L-缬氨酸(L-valine) C5H11NO2 0.84 0.13 7 21.11 1.07 L-酪氨酸(L-tyrosine) C9H11NO3 1.28 0.12 8 25.58 1.93 色氨酸(tryptophan) C11H12N2O2 0.71 0.61 9 10.63 1.69 糖代谢 磷酸(phosphoric acid) H3PO4 0.7 0.99 10 14.13 1.08 L-苹果酸(L-malic acid) C4H6O5 0.9 0.64 11 17.03 1.56 L-苏糖酸(L-threonic acid) C4H8O5 0.81 0.37 12 20.1 1.63 柠檬酸(citric acid) C6H8O7 0.77 1.99 13 20.91 4.44 D-果糖(D-fructose) C7H15NO6 0.82 0.68 14 21.38 1.34 D-葡萄糖(D-glucose) C6H12O6 1.33 0.68 15 22.3 1.91 松二糖(turanose) C12H22O11 0.81 0.66 16 24.27 2.26 肌醇(inositol) C6H12O6 1.34 0.59 -
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